Thomas C. Mitchell

The Vaccine Adjuvant Monophosphoryl Lipid A as a TRIF-Biased Agonist of TLR4

The inflammatory toxicity of lipopolysaccharide (LPS), a component of bacterial cell walls, is driven by the adaptor proteins myeloid differentiation factor 88 (MyD88) and Toll-interleukin 1 receptor domain–containing adapter inducing interferon-β (TRIF), which together mediate signaling by the endotoxin receptor Toll-like receptor 4 (TLR4). Monophosphoryl lipid A (MPLA) is a low-toxicity derivative of LPS with useful immunostimulatory properties, which is nearing regulatory approval for use as a human vaccine adjuvant. We report here that, in mice, the low toxicity of MPLAs adjuvant function is associated with a bias toward TRIF signaling, which we suggest is likely caused by the active suppression, rather than passive loss, of proinflammatory activity of this LPS derivative. This finding may have important implications for the development of future vaccine adjuvants.

Activated T cell death in vivo mediated by proapoptotic bcl-2 family member bim.

At the end of the T cell response, the majority of the activated T cells die. We activated Vbeta8(+) T cells with staphylococcal enterotoxin B (SEB) in vivo and monitored the expansion and deletion of Vbeta8(+) T cells. We found that, in response to SEB, activated T cells died in vivo in the absence of Fas or TNF-R signaling but not when they overexpressed human Bcl-2. We also found that Vbeta8(+) T cells from Bim-deficient mice are resistant to SEB-induced deletion. While Bim levels did not change, endogenous Bcl-2 levels within Vbeta8(+) T cells decrease following SEB injection. Thus, the death of superantigen-stimulated T cells in vivo is mediated by Bim and may be modulated by a decrease in Bcl-2.

Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant

Abstract.The development of non-infectious subunit vaccines greatly increases the safety of prophylactic immunization, but also reinforces the need for a new generation of immunostimulatory adjuvants. Because adverse effects are a paramount concern in prophylactic immunization, few new adjuvants have received approval for use anywhere in the developed world. The vaccine adjuvant monophosphoryl lipid A is a detoxified form of the endotoxin lipopolysaccharide, and is among the first of a new generation of Toll-like receptor agonists likely to be used as vaccine adjuvants on a mass scale in human populations. Much remains to be learned about this compound’s mechanism of action, but recent developments have made clear that it is unlikely to be simply a weak version of lipopolysaccharide. Instead, monophosphoryl lipid A’s structure seems to have fortuitously retained several functions needed for stimulation of adaptive immune responses, while shedding those associated with pro-inflammatory side effects.

Immunological adjuvants promote activated T cell survival via induction of Bcl-3

Injection of soluble protein antigen into animals causes abortive proliferation of the responding T cells. Immunological adjuvants boost T cell responses at least in part by increasing the survival of activated T cells during and after the initial proliferative phase of their clonal expansion. To understand how adjuvants promote T cell survival, we used gene microarrays to analyze gene expression in T cells activated either with antigen alone or in the presence of two different adjuvants. Among the genes whose expression was increased by both adjuvants was the IκB family member Bcl-3. Retroviral infection experiments showed that expression of Bcl-3 increased survival of activated T cells in vitro and in vivo. Adjuvants may therefore improve survival of activated T cells via induction of Bcl-3.

The low‐toxicity versions of LPS, MPL® adjuvant and RC529, are efficient adjuvants for CD4+ T cells

Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less‐toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long‐term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon‐γ following a 21‐day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long‐term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low‐inflammation adjuvants, such as MPL and RC529, are capable of enhancing short‐term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long‐term retention of antigen‐specific T cells.

T cell apoptosis and reactive oxygen species

Summary Thousands of studies of ROS and their effects on cellshave been reported. Therefore it is not surprising thatthese powerful biological mediators play a role in thephenomena associated with T cell activation and death.Even though activated T cells can die in two differentways, driven by external stimuli via AICD or by process-es that are intrinsic to the cell via ACAD, evidence sug-gests that ROS are involved in both pathways.Although much of the signaling mediated by ROS in Tcells remains unclear, the abundance of data on ROSsignaling in nonlymphoid cell types should aid in theelucidation of ROS-driven signaling within T cells. Fur-thermore, since ROS are so crucial in determining thefates of activated T cells, an understanding of how ROSachieve their effects may suggest therapeutic targets forthe destruction of autoimmune T cells, and for theimprovement of substandard vaccines — the twin goalsof modern applied immunology. Note added in proof . Reusch and colleagues recentlyshowed that ROS specifically downregulated CREB-dependent Bcl-2 promoter activity in primary neurons.(Pugazhenthi, S. et al. 2003. Oxidative stress-mediateddown-regulation of Bcl-2 promoter in hippocampalneurons.

IFN-β Production by TLR4-Stimulated Innate Immune Cells Is Negatively Regulated by GSK3-β

TLR 4 stimulation of innate immune cells induces a MyD88-independent signaling pathway that leads to the production of IFN-β. In this study, we demonstrate glycogen synthase kinase 3-β (GSK3-β) plays a fundamental role in this process. Suppression of GSK3-β activity by either pharmacological inhibition, small interfering RNA-mediated gene silencing, or ectopic expression of a kinase-dead GSK3-β mutant enhanced IFN-β production by TLR4-stimulated macrophages. Conversely, ectopic expression of a constitutively active GSK3-β mutant severely attenuated IFN-β production. GSK3-β was found to negatively control the cellular levels of the transcription factor c-Jun and its nuclear association with ATF-2. Small interfering RNA-mediated knockdown of c-Jun levels abrogated the ability of GSK3-β inhibition to augment IFN-β, demonstrating that the ability of GSK3 to control IFN-β production was due to its ability to regulate c-Jun levels. The ability of GSK3 inhibition to control IFN-β production was confirmed in vivo as mice treated with a GSK3 inhibitor exhibited enhanced systemic levels of IFN-β upon LPS challenge. These findings identify a novel regulatory pathway controlling IFN-β production by TLR4-stimulated innate immune cells.

Selective Activation of the p38 MAPK Pathway by Synthetic Monophosphoryl Lipid A

TLR4 stimulation by lipopolysaccharide can cause both MAL/MyD88- and TRAM/TRIF (Toll IL-1 receptor domain-containing adaptor-inducing IFNβ)-dependent signaling events. Monophosphoryl lipid A (MPLA), a low toxicity derivative of endotoxic lipopolysaccharide, enhances antibody responses, T cell expansion, and recall responses against antigens without causing excessive inflammatory side effects. Previously, we proposed that TRIF-biased activation of TLR4 by MPLA is responsible for its reduced toxicity while retaining potent adjuvant effects. However, some TRIF-associated genes, such as MCP-1, are only weakly expressed, and some MyD88-associated inflammatory and anti-inflammatory cytokines, such as tumor necrosis factor α and interleukin-10, are strongly activated after MPLA stimulation despite weak NF-κB but strong IRF3 activation. We now report that synthetic derivatives of MPLA retained TRIF bias as compared with synthetic diphosphoryl lipid A, indicating a change in a single phosphoryl group is sufficient for TRIF-biased TLR4 stimulation. We extend our previous observations by showing that sMLA induces strong p38 MAPK but weak JNK activation, resulting in high IP-10 (interferon-inducible protein 10), tumor necrosis factor α, and interleukin-10 but low MCP-1 transcript levels. Results of this study identify a novel biochemical mechanism for regulation of sMLA-induced gene expression.

Selective TRIF-dependent signaling by a synthetic toll-like receptor 4 agonist.

A single change in a synthetic lipid A mimetic may reduce the toxicity of this vaccine adjuvant. Biasing TLR4 Signaling Toward a Less Inflammatory Route Signaling downstream of Toll-like receptor 4 (TLR4) proceeds through two pathways that are dependent on distinct adaptor proteins. One of these pathways leads to immunostimulatory responses, whereas the other leads to the production of toxic proinflammatory cytokines. Compounds that could stimulate the beneficial arm rather than the proinflammatory arm would be useful as adjuvants for vaccines. Bowen et al. investigated two synthetic TLR4 agonists that differed only in their stereochemistry at a single point in their structure and found that whereas one agonist stimulated both arms of the TLR4 pathway, the other agonist stimulated only the noninflammatory branch. These findings have implications for the design of more effective vaccine adjuvants. In response to ligand binding to the Toll-like receptor 4 (TLR4) and myeloid differentiation-2 (MD-2) receptor complex, two major signaling pathways are activated that involve different adaptor proteins. One pathway depends on myeloid differentiation marker 88 (MyD88), which elicits proinflammatory responses, whereas the other depends on Toll–IL-1 receptor (TIR) domain–containing adaptor inducing interferon-β (TRIF), which elicits type I interferon production. Here, we showed that the TLR4 agonist and vaccine adjuvant CRX-547, a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic lipid A mimetics, displayed TRIF-selective signaling in human cells, which was dependent on a minor structural modification to the carboxyl bioisostere corresponding to the 1-phosphate group on most lipid A types. CRX-547 stimulated little or no activation of MyD88-dependent signaling molecules or cytokines, whereas its ability to activate the TRIF-dependent pathway was similar to that of a structurally related inflammatory AGP and of lipopolysaccharide from Salmonella minnesota. This TRIF-selective signaling response resulted in the production of substantially less of the proinflammatory mediators that are associated with MyD88 signaling, thereby potentially reducing toxicity and improving the therapeutic index of this synthetic TLR4 agonist and vaccine adjuvant.

Cutting Edge: Limiting Amounts of IL-7 Do Not Control Contraction of CD4+ T Cell Responses

During the acute T cell response most effector T cells die while some survive and become memory T cells. Selective expression of CD127 (IL-7Rα) on effector T cells has been proposed to engender their survival into the memory pool. We assessed the role of IL-7 in effector T cell survival using MHC class II tetramers to track a CD4+ T cell response following infection with a recombinant vaccinia virus (rVV-2W1S). Exogenous IL-7 prevented the contraction of the 2W1S-specific CD4+ T cell response after rVV-2W1S infection. IL-7 increased proliferation of, and Bcl-2 expression within, 2W1S-specific T cells; the latter was required for IL-7-driven prevention of contraction. Conversely, in vivo neutralization of IL-7 or Bcl-2 did not exacerbate the contraction of 2W1S-specific CD4+ T cells. These data suggest that IL-7 administration may enhance the survival of effector T cells but that IL-7 is not the limiting factor during normal contraction of the response.